PROJECT SUMMARY: Microglia/macrophage polarization after intracerebral hemorrhage Spontaneous intracerebral hemorrhage (ICH) causes high mortality and morbidity, but it is understudied compared to ischemic stroke, and effective treatment is lacking. Hematoma volume and expansion are independently associated with poor patient outcomes; therefore, rapid removal of toxic blood could limit ICH- induced brain injury. After ICH, microglia and macrophages (MM?) shift activity states to remove toxic blood and may protect the brain. However, over-activated MM? cause secondary brain damage by releasing cytotoxic substances. These opposing effects may result from distinct MM? subsets, which are categorized into classically activated proinflammatory (M1) and alternatively activated anti-inflammatory (M2) cells. Alternatively activated M2 MM? exhibit increased phagocytosis of apoptotic cells, which could involve activation of the scavenger receptor CD36 and inhibition of its negative regulator toll-like receptor (TLR)4. Additionally, interleukin-10 (IL- 10), an anti-inflammatory cytokine, represses inflammation, polarizes macrophages to an M2c subtype, and enhances phagocytosis. High levels of IL-10 in blood or brain tissue have been reported in ICH patients. However, MM? polarization and the exact role of IL-10 signaling in M2 polarization after ICH are unknown. The long-term goal of our research is to limit ICH injury and improve functional outcomes. The overall objective of this R01 is to investigate whether modulation of MM? phenotype and function by IL-10 reduces ICH injury and improves histologic and functional outcomes. Our preliminary studies showed that IL-10 expression increases in brain slice cultures exposed to hemoglobin and in an in vivo model of ICH; that IL-10 increases microglial phagocytosis and CD36 expression in brain slice cultures; that IL-10-deficient mice have impaired hematoma resolution and altered CD36 and TLR4 expression compared with that in C57BL/6 wild-type mice; and that exogenous IL-10 successfully reduces hematoma volume. These findings prompt the hypothesis that polarizing MM? to M2 phenotype by IL-10 reduces ICH injury and improves histologic and functional recovery after ICH. In three specific aims, we will determine whether M2 microglial polarization by IL-10 is responsible for phagocytosis in ex vivo brain slice cultures exposed to hemoglobin or aged red blood cells (Aim 1); whether M2 MM? polarization by IL-10 improves the histologic and functional outcomes after ICH in vivo (Aim 2); and whether IL-10-induced MM? M2 polarization requires activation of CD36 and inhibition of TLR4. The information gained from this study will provide us with novel insight into the MM? polarization after ICH and the cellular and molecular mechanisms by which IL-10 signaling?induced MM? M2 polarization reduces ICH injury. Based on multidisciplinary approaches, our findings will potentially lead to a new therapeutic strategy not only for ICH but also for other brain disorders. This novel proof-of-concept work to study modulation of innate inflammation is a critical priority identified by the recent NINDS-SPRG.